JPS63500001A - Transfer of male sterility in carrots - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Transfer of male sterility in carrots Background of the invention field of invention The present invention relates to carrot (Daucus strain) which produces hybrid seeds. used for For more information, see Δ The present invention enables plant breeders to commercialize the desirable trait of male cytoplasmic sterility (CMS). ; making it possible to incorporate this into desirable carrot varieties.

Normal flowers are self-pollinating and sib-pollinating, so male sterility is a problem in carrot hybrids. It is valuable in breeding offspring. Male sterile carrot lines produce vigorous pollen and cannot self-pollinate. By eliminating the pollen of one parent variety in the cross This ensures that plant breeders obtain hybrid seeds of uniform quality. Currently, the cytoplasm Male sterility is not easily obtained in all carrot varieties; Growers should not rely on traditional selective breeding to incorporate CMS into new carrot lines. Must be.

The invention described herein provides commercially desirable maintenance of CMS strain protoplasts. Modern biology hybridizes with either isolated nuclei or protoplasts from carrier strains Including the use of technology. Cytoplasm of CMS cells with enucleated nuclei of male fertile carrot cells This allogenic cell can then be regenerated by inserting it into a mature plant. This allows carrots to possess both cytoplasmic male sterility and desirable commercial properties. A plant body is obtained. These plants were then used as parent lines and subjected to standard selection. Grow new carrot hybrids using selective breeding techniques.

In addition to the above-mentioned method, the invention also provides the product of said method, i.e. male sterile carrots. A novel lineage of protoplasts and hybrids regenerated therefrom is described.

Disclosure of information CMS Carrots are divided into two groups, petaloid and brown-rot-type male sterile. It has been established through previous research that it can be classified. In petal-like CMS, the effect is The structure of the affected plant is abnormal and resembles flower petals. Thompson D. Thompson D, J.

), Studies on the Inheritance on Mail Sterility Studies on the Inheritance of Male 5terility in the Carrot), a American Society for Cultural Science (Ame) r, Soc.

Hort, 5ci-), 78: 332-338 (1961). brown In -rot-type male sterility, the pollen is present but turns brown and does not produce pollen. to save. Welch, Joy E. and Grimball, E.L. , J, E.

and Grimball, E, L,), Male Sterility in... Carrots (Male 5terility in Carrots), Sai 5science, 106: 594 (1947).

The value of producing male sterile parent lines for use in hybrid seed production is known. There have been no successful studies to date to fuse tobacco protoplasts with CMS. It is stated in. Zelcer A-.

et at, ), interspicy transfer on site Rathmink mail sterility pie fusion between pro utplus on normal niktotiana sylvestris and ed Kuslay Irradiated Prout Plus on Mail Ste Lyle NΦTabacum (Interspecific transfer o fcytoplasmic male 5terility by f+rsi on between protoplasts of normal N1ct otiana 5ylvestrisand x-ray 1rradiate d protoplasts of male-sterile N, tab acum), Zeitschrift Viel Franzen Physiologie (Z.

Pflanzenphysio+, Volume 90.3.397-407.1978 ゜To the best of the applicant's knowledge, there is no fusion of two carrot cells between carrot varieties or No one has ever reported successful insertion of an allogenic nucleus. However, carrots A full-blown fusion was reported between the Umbilifera polyphylla (of which N is a member). There is. Dutits, D., et al. Intergeneric Gene Transfer Medium Rad Pi ・Plant Proudoplast Fusion (Intergeneric g ene transfer mediated byplantprotopl ast fusion), Molecular and General Genetics Cus (Mo1e, Gen-Genet,) 179: 283-288.1 The method for producing protoplasts for carrots by adding 980° also includes protoplasts. Methods are also known for regeneration in which plasts are introduced into mature plants. Ste Ward, F.C. et al. (Stewart, F.C., et al.) Investigations on Growth and Morphogenesis in Cal Observations on gro wth and morphogenesis 1ncuHured cell of carrot) (Daucus ca roia L,))% Philosophical Transactions on Royal Society on London Selby (Ph1los, Tr. ans, R.

Soc, London Ser, B), 273: 33-53, 1 980゜Grambow N, J,, eta l,), Cell Division and Plant Development from ・Protoplast on carrot cell suspension culture yard ( Ce1l division and plant development fromprotoplasts of carrot cell 5 uspe culture).

Plants (Berl, ) 103: 348-355 ,972O Summary of the invention The present invention relates to the application of modern biology to the breeding of new carrot varieties. CMS d By fusing carrot protoplasts with a desirable carrot variety, a second Desirable male sterile carrots that produce hybrid carrot seeds when crossed with lines of It becomes possible to produce a parent strain that

The method described herein transfers intact nuclei of maintainer cultivars to CMS carrot cultivars. into nucleated protoplasts or nucleated protoplasts of maintenance cultivars into C. Either by direct fusion with a functionally enucleated protoplast with MS characteristics. This includes the introduction of a cytoplasmic male sterility trait into the carrot maintenance line.

The nucleated protoplasts are optionally treated with a substance such as iodoacetic acid or rhodamine 6-G. May be inhibited with cytoplasmic inhibitors. Next, the fused protoplasts are introduced into the plant body. It is then maintained through traditional breeding methods to produce parental lines for hybrid seed production. Used as a standard.

New CMS parental lines of carrot using allogenic nuclear insertion or hybrid cell fusion to produce. The advantage lies in time savings by avoiding traditional selective breeding techniques. Ru. Selective breeding to introduce CMS into desired carrot lines typically involves io/ , takes 1" to 12 years. Cell fusion can achieve the same results within 4 to 5 years. Wear. time (plus the space and manpower required to raise successive generations). The quantity is also greatly reduced by these new methods.

In addition to the methods described herein, male sterile cultivars with nuclei from maintainer cultivars A novel product consisting of allogenic protoplasts of the ginseng variety has also been described.

Finally, herein we discuss cells regenerated from allogenic protoplasts and hybrid varieties. is listed.

[The term cytoplasmic male sterility J (CMS) refers to both forms of sterility, i.e. petaloid and brown-decay-type CMS.

The phrase ``allogenic rho protoplast'' refers to the nucleus of a plant that is enucleated and then immediately asked. Another word is ``allogeneic'', as in the case of protoplasts that have been forcibly accepted. An organism that has or contains the nucleus of an organism. allogeneic protoplast Streptococcus is produced by nuclear insertion or by fusion of protoplasts into protoplasts. You can germinate it.

The word "cell" refers to a living plant material consisting of an intact cytoplasm, nucleus and cell wall. Say the unit of number.

The term "enucleated" refers to a cell with a non-functional, vulnerable nucleus. this is achieved by X-ray processing.

The phrase "significant amount of cytoplasm" is stable in allogenic cells for successive generations. Refers to the amount of cytoplasm sufficient to confer l'cMs properties.

Detailed explanation The following detailed description of the different varieties of Daucus carota is given in a non-limiting manner. It should be understood that this includes steps that may vary. The determination of those conditions is is within the scope of ordinary skill in the art.

The invention disclosed herein is applied to Michigan State University (MSU) 5986A or Many publicly available cytoplasmic male sterile carrot lines such as MSU 1302A It can be implemented using either of the following. There is no superiority or inferiority among the known strains. Appropriate ra' In addition to selecting cMs strains, the availability of desirable maintainers is limited. . The maintainer line transfers the function to the male pore part of the second type when crossed with the CMS line. It is a physically normal carrot variety that does not allow regeneration. The maintainer line is the new variety. When choosing an appropriate maintenance strain, select the desired physical characteristics. Gender is also considered.

For CMS strains, suitable maintenance strains are known.

Determination of alternative maintenance lines involves simple crosses between CMS lines and various male fertile lines. This is achieved by continuing until a sustainer is found. The following systems are CMS and its An example of a corresponding maintainer strain is: (1) Michigan State University (MSU) variant Seed Funeral Car 1209 MS and USDA: (3) MSU 1302A and and MSU 1302B: and.

(4) MSU 5931S and MSU5931M0S as all 5931M stocks Potential alternative CMS lines are available to those familiar with the field of carrot breeding. It is possible. State and Federal Agricultural Experiment Stations agricultural experiment 5tation) continues We were able to produce a new CMS carrot and are still producing it. Learn more For example, CMS carrot varieties and their maintenance lines are USDA carrot and Onion Improvement Program, Wl., Madison, U.S.C. Available through the University of New York, USDA/Horticulture. Alternative sources may be e.g. , Gainesville, Florida, University of Florida: College Station, Texas (CoCo11e5tation).

Texas A&M: and Idaho State, Moscow, and University of Idaho Cooperative State Q-Technology Laboratory (Cooperative 5tate Ag) ricultural Experiment Station).

Once the CMS and maintainer line selection is complete.

Seeds of the desired strain are germinated to produce source material for protoplasts. Extinction Seeds by soaking in a fungal solution, preferably dilute sodium hypochlorite surface sterilized at an appropriate temperature, preferably 25°C.

Germinate in a dark, moist environment. The seedlings are used to produce a callus culture, and protoplasts are obtained.

A callus culture starts from the root segment of the seedling. A few days after germination, the segment Basic ten GR containing salts, carbon sources and growth regulators (GR) Place on agar medium to maintain growth. The basal medium is a complete medium that maintains the culture of plant cells. It is a minimal medium containing balanced nutrients. The preferred basal medium is: Examples are provided, but alternative basal media are known to those skilled in the art. basal medium The presence of growth regulators in is indicated by GR. 15-32℃, preferably Incubate the tissue in the dark at a temperature of 26-28°C for either an incubation phenotype or a white phenotype. Obtained from a CMS carrot plant having any of the following. The root is sodium hypochlorite First surface sterilize in a sterile solution such as sterile solution. Small, about 1 to 4 cubic millimeters Discs are cut from the cambium area and placed on basal 10 GR agar. Then the corresponding Incubate the root segments in the dark at 15-32°C, preferably 26-28°C. start.

After about 4 to 8 weeks, callus tissue from either root segments or cambial parts Create a subculture medium. A small piece of callus tissue with an initial weight of 0.1 to 0.3 g Samples were transferred to basal agar 16GR medium and incubated at the same temperature under cool white fluorescent light for 16 minutes per day. Incubate for an hour. Cultivate by passage every two months under similar conditions. You can maintain the save. The callus tissue is then used to produce suspension cultures, and then Cell material for protoplast production is obtained.

Approximately 0.1 to 0.3 g of callus tissue was placed in a basal 1GR liquid medium 2 in a suitable flask. Start suspension culture by transferring into 5 mQ. Under cool white fluorescent light, 25-28℃ The culture is incubated on a horizontal rotary shaker.

Suspension 5 mQ of fresh basal + GR liquid medium 25 n in a suitably sized flask By transferring to +f, suspension cultures can be passaged every 12-16 days.

The suspension is used as a basal medium, cellulase, and pectinase.

and a protoplast isolation medium containing a suitable osmotic agent such as mannitol. Protoplasts are prepared from the cell suspension by mixing with soil. to 30℃ Incubate for 3-4 hours on a rotary gyrator in the dark. Afterwards, passing through a screen and/or Lymphopr. Osmotic pressure such as Nygard (A/S, Norway) target; protoplast migration through or onto a stable density cushion. Separate released protoplasts from cell masses and other foreign materials by cardiodissociation . The above-mentioned technique is well known and is described by Larkin, P.G. J,) C Planta (Berl).

), 128:213-216.1976) ing.

Protoplasts are being formed in the breeding method for handling protoplasts. It is necessary to check the quality and quantity of the samples. Such methods allow determination of cellular activity, residual determination of cell wall material and simple counting of protoplasts. disclosed The specific methods used in the method are described in the Examples below. Alternatives to equal effects The method is from The Handbook on Plant Cell Culture (the Han dbook of Plant Ce1l Culture), Volume 1, Techue Techniq for Propagation and Breeding (Techniq) ues for Propagation and Breeding), Do8day0A Evans, W.R. Sharp, P.B.A. Millard and Y. Yamada (Ed, D, A.

Evans, W, R, 5harp IIP, V, Amm1rato an d Y, Yamada), Macmillan Dekashisha, New - York.

124-177, (1983), on protoplast research. Found in general literature.

After determining quality and quantity, repeat low speed centrifugation (700-800G) and resuspension. Wash the protoplast culture three times in wash medium by washing. Washed culture The substrate is a mixture of a basal medium and a suitable osmotic agent such as mannitol.

Introducing nuclei from the maintainer line into protoplasts from the CMS line requires isolation. Insertion of nucleus into enucleated CMS protoplasts or removal of maintainer protoplasts Actual fusion into nucleated male sterile protoplasts.

This can be achieved by A preferred method is to maintain protoplasts with cytoplasmic inhibitors. The X-ray treated protoplasts from the CMS strain were transferred to the maintenance strain. The goal is to fuse it with protoplasts.

To isolate nuclei from maintainer cultivars, protoplasts are suspended in basal medium and incubated at low speed. in 2% gum arabic pelleted by centrifugation and dissolved in nuclear stabilization buffer. Resuspend in Nuclear stabilization buffers can maintain intact nuclei, and in the literature It is publicly known. The specific M solution used herein is designated R3 and is , Cress, D, E, et al., Plan ta), 143: 241-253.1978, and the following examples. ) is described in detail here. The protoplasts were then subjected to French pressure braking. Place in French press. About 1000 Slowly increase pressure to 2500 psi, followed by slow release. rupture the protoplasts by

It is then collected aseptically.

The ruptured protoplast solution was mixed with RS buffer and a substantially larger (10 %) over a nuclear isolation buffer consisting of gum arabic. Then, the nucleus is calmed down. Dissolve the two species with enough force to pellet them but release other organelles into suspension. Centrifuge the liquid. The nuclei were resuspended in 2% gum arabic in R5 buffer and further Remove remaining cellular foreign material by centrifugation and resuspension twice. final core The pellet is resuspended in a small amount of 2% gum arabic in R3 buffer.

Although nuclei obtained from protoplasts are preferred, nuclei are also protoplasts. It can also be obtained from cells or cell suspensions rather than from cells. Basic 10 GR Centrifuge the cell suspension in liquid medium to pellet the cells. Then, R3's The cells are resuspended in 2% gum arabic in a suitable nuclear stabilization buffer such as. next Then, place the cells in a French pressure breath and apply a pressure of 2000 to 4000 psi. rupture by slowly releasing. The ruptured cells are then placed in a small tube. Sieve through N1tex or a uniform screen continuously. Poppy seeds, the resulting organelles were layered on a single layer of 10% gum arabic gradient, and the nucleus Spin with moderate force to gently pellet the cells and leave other organelles in suspension. Turn around.

CMS Protoplasts from carrot lines must be enucleated prior to hybridization. Must be. The ability of the nucleus to divide and regulate the cell while preserving the functions of the cytoplasm By exposing the protoplasts to high-voltage X-rays at levels that effectively destroy the enucleation is achieved. Enucleate CMS carrot protoplasts; this is an effective The total line l can be any one from 5 kiloroentgen [kr] depending on the product used. It is in the range of 100 kr.

To determine the appropriate X-ray dose range, kill 100% of the protoplasts. The minimum level of X-ray exposure must be determined. Percent death regenerate cells Determine by placing in culture medium and counting colony formation. 100% death In order to obtain the most accurate x-ray dose, use a cell feeder and a controlled cell suspension. It is preferable to use a free cell culture medium or a suitable low density cell rescue method. Optimize the conditions for growing small populations of cells that are not resistant. Kills 100% of cells When determining the minimum dose required to achieve The protoplasts are exposed to 10 and 20 kr above and below the minimum dose. each Protoplasts from exposure levels are used for hybridization. Cells are exposed immediately to X-rays. Used later.

As mentioned above, re-introduction of functionally enucleated protoplasts can be carried out using isolated nuclei or This can be achieved either with intact protoplasts.

To insert isolated nuclei into protoplasts, immediately after exposure to X-rays, functional The enucleated protoplasts are collected and mixed with a 5- to 10-fold excess number of nuclei.

The mixture is centrifuged and resuspended in wash medium. This mixture promotes nuclear uptake. The accelerating fusogen is added through the protoplast cell membrane. Such a fusion inducer is well known and is published in the Handbook on Plant Cell Culture (Handb). ook of Plant Ce1lCulture).

cit., pp. 291-321. Preferred buffers are Chao and Kao & Michayluk, Planta (Berl, ) 115: 355-367゜Polyethylene described by 1974 Recall (PEG) fusion method is utilized.

Fusion of protoplasts into protoplasts is a preferred method. These methods avoids the need to extract intact nuclei. cells to be satisfied about carrots There are two ways to achieve fusion. The most preferred method is to use cytoplasmic inhibitors. Ru.

In the first method, the fusion of enucleated and nucleated protoplasts , E (Galum, E).

Tumatic Cell Fusion for Inducing Cytoplus Mink Exchange (Somatic Ce1l Fusion for Inducing Cytoplasmic Exchange): A. New Biological System for Cytoplus Mink Genity x in buyer planta, in plant in bluetonto a New Biologics (A New Biologics) al System for Cytoplasmic Genetics in Higher Plants, InPlant Improve, and Somatic Ce1l Genetics), Nido I.K. Basil (Ed. 1., Vasil et al.

) 2 Academic Publishers, pages 205-220 This is accomplished using a PEG-containing fusion buffer as described. intact protoplastic If fusion between cells is used, successful transfer of CMS properties will occur during subsequent cell division. depends on complete separation of cytoplasmic material. Due to the possibility of fragmentation, a large number of reproduction images are The success of this method must be tested in field trials.

In the most preferred method, the cytoplasm of the sustainer protoplasts contains citrob, citrob, and V. A. et al., Chloroplast. Transfer in Nicotiana Baste on Metapolisk Comple MENTION BETWEEN ERADIENTED AND IOTOR Chloroplast Tra nsferin N1cotiana based on Metabolic Complementation between Irradiated a nd Iodoacetate Treated Protoplasts), Planta, 152: 341-345, 1981: and Ziegler and Davids on), Illumination on Mytochondral Elements INDIVIDUAL BUT ABILITY ON HYBRID CELLS, TUMATÉ Illumination of Mito chondral Elements and Improved Viabi lity of Hybrid Cells, SomaticCell Gen etics), vol. 7, pp. 1.73-88. treatment with cytoplasmic inhibitors such as iodoacetic acid or rhodamine 6-G. It is suppressed by this.

To determine the optimal dose of cytosuppressant for a particular breed, consider the susceptibility range. must be obtained. As with X-ray irradiated cells, low cell density rescue It may be desirable to determine optimal killing levels of quality inhibitors.

Due to the inherent variation in the susceptibility of protoplasts to treatments, there is a range of CMS protoplasts and auditory protoplasts with faX-rays or cytoplasmic inhibitors need to be processed. For any particular protoplast preparation, 10 The minimum amount of X-ray dose or exposure to cytoplasmic inhibitors that achieves 0% mortality. It is important to make accurate predictions. Therefore, the product treated with multiple levels of X-rays Fusion of toplasts and cytoplasmically suppressed protoplasts is due to biochemical complementarity. It is based on a matrix experimental design method that best represents the level of inhibition.

Maintainer protoplasts are exposed to various exposures to cytoplasmic inhibitors and CMS exposed to qt X-ray irradiation; matrix fused with protoplasts By using experimental design, we can select the population with the maximum number of allogenic cells. Ru.

This includes chemically suppressed and X-rayed cells from each test prior to fusion. This is achieved by sampling. Has recovered activity after fusion A population that does not have activity before fusion is presumed to be allogenic cells.

Fusion with X-ray treated cultures was performed as described above for non-cytoplasmically suppressed protoplasts. Achieved by sea urchin. Forced selection on allobiotic protoplasts leads to non-fused cells The number of colonies and plants selected for on-farm trials is substantially reduced. This method is preferred because it

Regeneration of allogenic protoplasts into multicellular colonies is completely balanced. in a regeneration medium containing salts, multiple carbon sources, osmolytes and growth stimulants. It happens in Several types of media promote protoplast regeneration for many plant species. It is known that

Each medium must be tested for its ability to regenerate the specific protoplasts used. No. Such experimental work is well within the capabilities of those skilled in the art. preferred The regeneration medium is designated A&S and is described in detail in the Examples below. allogeneic cells Regeneration optionally feeder layer, conditioned media or alginate encapsulation This may be facilitated by the use of low density cell culture methods such as.

Fused protoplasts growing in regeneration medium rapidly recover their cell walls and Form microscopic colonies within weeks. Replace the medium every week and after 3-4 weeks , plate the colonies and place them on agar containing embryo basal medium. promotes embryogenesis and contains fully balanced nutrients, but in regeneration medium Contains no carbon sources found. Preferred embryo media culture callus tissue The basal medium is the same as that used for this purpose, but without the growth regulators. form a colony Remove the embryos and place them in a tube containing basal agar.

Once they reach a suitable size, the seedlings are transferred to soil and grown in a mist chamber. . Established plants are vernalized to force flowering, and maintenance lines, preferably nuclear sources, are Pollinate with the same strain that you will be offering. For the production of hybrid seeds, the plant body is Conventional breeding techniques known in the art such as fertilization with pollen from a second desired variety The stability of CMS and other desired horticultural properties of the seeds obtained using the technique test.

Example The detailed rl examples below explain how to prepare Di Carota's allogenic protoplasts. and describes how to carry out the various methods of the invention. The example simply are intended to be illustrative and not to be construed as limiting the foregoing disclosure. Should. Those skilled in the art will be familiar with the contents of the medium, growth conditions, and other techniques. One will readily recognize suitable variations from the law.

culture, callus and cells A, culture medium The basal medium used to culture callus tissue is Murashige and Skoog (Mura shige and Skoog) [Physiologia plantarum (P) Iysiol, Plant, ) 15: 473-497.1962) salt formulation and varnish sucrose containing the following in my/l: 2 0000; Thiazine HCI, 0.1: i-inositol, 100; Ni Cotinic acid, 0.5: Pyridoxine/HCI, 0.1. ri lj resin 3.0゜ group In addition to the components in the basal medium, the basal agar medium contains the growth regulator 2,4-dichlorophyll. Phenoxyacetic acid (2,4-D), 1.0m? / mQ: and kinetin, 0 , 2'F/rnQ. The agar medium was 7 g/l Sigma (Sigma ) Contains agar. pH of the medium prior to autoclaving or addition of agar Adjust to 5.5.

Prepare the liquid medium with SOmQ and use 125 company's Delong flasks. Put it in. Prepare agar medium.

After melting, 20md! / Tube capacity: 25 x 150 tubes It will be done. Cap the tube and flask and incubate at 121°C.1. osKL9/cd Autoclave for 15 minutes.

B. Start of callus and suspension culture. Di Carota, Michigan State University variety #Car with cytoplasmic male sterility characteristics 1209MS and maintenance system VSDA 6274n with 10% sodium hypochlorite. Sterilize by immersion in thorium solution for 15 minutes and damp sterilize at 25 °C. Germinate on Okigami in a dark place. To start the callus, after 5 to 7 days, root segment Cut out the material, place it on a basal 10GR agar medium, and incubate it in the dark with a trowel at 26-28°C. Incubate.

After 6 weeks, callus tissue with an initial weight of approximately 0.2 g was cut from the root segment and basal Passage to 10GR agar medium and ink for 16 hours per day at 25°C under cold white fluorescent light. cuveate. Passage approximately 0.2 g of tissue onto basal 10GR agar medium every 2 months. By maintaining a callus preservation culture.

Approximately 0.2 g of callus tissue was placed in a 125 mQ delong flask. Scochu O Basic Ten GR Liquid Medium 50ml! Open suspension cultures by passage in start A horizontal swivel machine set at 150 rpm at 25°C under cool white fluorescent light. <Model G-2, Ninie Brunswicz Scientific (New) Brunswick (5 scientific), Ninny Brunswick ( Incubate the culture on the New Brunswick, New Jersey) Bate. Add 5 mQ of cell suspension to a new basal 10 G flask in 12 SrnQ Subculture the suspension culture every 14 days by subculturing into 50 mQ of R liquid medium. do.

■Isolation of protoplasts Pellet the cell suspension by centrifugation and add basal medium and 0.56i1 man Resuspend in wash medium consisting of nitol. 0.56M man as an osmotic pressure regulator 2.0% Cellulase R-10.2 in basal medium pH 5.8 with nitol. Enzyme solution containing 0% cellulase RS and 1.0% pectolyase Y23. Add an equal volume of the solution to the cell suspension in wash medium. 75 rpm orbital shaker Incubate the cell suspension in the presence of enzymes at 28-30°C. . After 1 hour, the first protoplasts were released, and after 3-4 hours they were placed in the enzyme solution. The process is complete. Somewhat bulky cell clumps never produce protoplasts and are %25 μm thick. It is removed by passing it offshore through a tsushu. Crude protoplast suspension 1 0 mQ on top of a 5 m flood of Limhoprep in a 15 mf centrifuge tube, ~200 Further purification is performed by spinning at XG for 10 minutes. pasteur pipette Collect purified protoplasts from the medium-limhoprep interface. 100xG After washing three times with wash medium for 5 min at is ready for other operations.

■, Preliminary procedure A for protoplast isolation, measurement of cell activity Protoplast activity was determined by Widholm's fluorescein Acetate method (5tain Technology), 47: 189-194.

(1972). 5η/rnQ acetone dissolved stock solution of dye Fluorescein diacetate staining by dilution in protoplast culture medium Prepare a color solution (0.01%). This preparation can only be used for a few hours and It must be created separately. Mix equal amounts of staining solution and cell suspension Test protoplast activity by. After 5 minutes, place the cells in a hemocytometer and excite filter, 330-500μ, and cut-off filter, )460nm. Lymphus Vanox (Olympus Vanox) Bell. Living cells emit yellow to green fluorescence; dead cells do not emit fluorescence. .

B. Measurement of cell wall IJ - Quit Calcol Fluor White (quid Calcol Fluor White) fluor White) ST (American cyanamide) n Cyanamid), Wayne, New Chassis State, USA) was diluted 100 times with protoplast culture medium to suspend the protoplasts. Mix with equal amounts of liquid. After a few minutes, replace the same filter set up for the activity measurement. Examine the cells under a fluorescence microscope with a router. Only cells with undigested cell walls are fluorescent shows.

Counting of C1 cells Quantitative evaluation of protoplasts was carried out by Fuchs-Rosental. (enthal) Performed using a hemocytometer.

■, plating and culturing protoplasts at a concentration of 0.5-1.0×105 cells /mQ isolated protoplasts were plated in regeneration medium and incubated at 25°C. Place in dark growth chamber A for 5-7 days. The regeneration medium is 150 m from the basal medium? /l D-xylose, 150Hi//D-arabinose, 1oomg7t Lucose.

34 g/! Sucrose, 45.5 g/l sorbitol.

0.01η/l dimethylallyladenine and o,2my/12.4-dichloro Contains phenoxyacetic acid (pH 5,8). The culture is then exposed to low amounts of indirect light. Moving.

■, Isolation of Nantes nucleus A, Protoplast as the source material Isolated protoplasts were incubated overnight at high density in protoplast culture medium. Bate.

Rotate the protoplasts for 5 minutes at 700XG, add 0.4M sucrose, 5mM 2, N-morpholinoethanesulfonic acid, 50 μg/mQ DTT DL-di Thiothreitol, 0.15% 2-ethylhexanol% 6mM acetic acid Si, 25mM potassium chloride (pH 6,4) and 0.5% dimethyl sulfonate Resuspend in 2% gum arabic in R8 buffer containing oxide for 2 hours at room temperature. Incubate. Protoplasts are processed using a French pressure cell press (French pressure cell press). pressure cell press) (American instrument American Instrument, Silver Spring (5ilver Springs, Maryland);

Protoplasts do not need to be adjusted to any particular density. rubber stopper Insert a sterile 50 mρ plastic centrifuge tube with a The protoplasts are collected aseptically. To rupture protoplasts, pressurize Slowly increase the force to 2000 p'si and then release into the containment tube. . Samples of ruptured protoplasts containing intact nuclei are examined under a microscope to determine optimal rupture. Determine the crack conditions.

15ml of 5mQ of ruptured protoplast solution! Falcon ) on 10m2 of 10% gum arabic in R5 buffer in a plastic centrifuge tube and centrifuge at 700xG for 20 minutes. 2% gum arabic in R5 buffer Resuspend the pellet in the medium and centrifuge for 5 min at 700xG to remove the cytoplasmic material free of nuclei. Clean the quality. Repeat this last step three times. 2% arabic final nuclear pellet Resuspend in 2 mQ of R5 buffer containing Agam.

A 100 μA' sample of nuclei was added to a Hoechst #33342 (x ,og/I! Stain with 50 μl of stock solution and analyze with Fuchs-Rosenthal hemocytometer. Count with . Pellet and resuspend the nuclei in wash medium prior to use in fusion. Ru.

B. Cell suspension as source material The established cell suspension is refined by centrifugation.

Discard the culture medium and resuspend the cells in 2% acacia in R3 buffer. Hula Place the cells in a pressure cell press and rupture them at 2-4000 psi. dissolve The cells 117.61, 43, 25.15. and 10 μ of Nitex (N1 tex) Sieve through a screen, 11, 15. and 20% Arabi Layer on top of the Agum viscosity step gradient and centrifuge at 900XG for 12 minutes.

The pellet was nucleated by staining in Hohest tk33342 as described above. Check for purity.

■Preparation of CMS cytoplasm by X-ray irradiation of protoplasts Prototypes were prepared from established CMS callus cultures of MSU 1209M5 as described above. Plasts are isolated and suspended in wash medium. After cell counting and activity testing, Philips Electronique India)') - (PhilipsEl ektronik Industry) GMBH (Mburg, Germany) A large number of protoplasts were irradiated by an MG-301 high-voltage X-ray device manufactured by M. Co., Ltd. messenger Before each use, use a Victoreen probe. , (5helter-Globe), Pictrine Equipment Division, Cleveland (C1eveland), Ono, Io. Standardize line equipment.

Protoplasts 10.13.16. and exposure to changes in 19 kiloroentgen do. Protoplasts from each exposure are used in the hybridization method described below.

■Hybrid formation A. Polyethylene glycol using CMS uptake of isolated nuclei by protoplasts The PEG fusion method is described by Kao and Mich. ayluk) C Planta (Berl, ), 115 : 355-367.

The method described by (1974) was modified. Treated with X-rays in washing medium Mix protoplasts and isolated nuclei in a numerical ratio of 1=5 or 1:10 to create a suspension. Obtain a final protoplast concentration of 2 x 10 cells in 0.5 mQ. 22.5 %PEG6000 (Sigma).

10 m M Ca Cl 2 and 4% sucrose (pH 5,8-6.0) Add 2 mQ of PEG solution to the protoplast mixture, followed by gentle mixing. Combine and incubate for 10 minutes at room temperature. Basal medium and 0.56M mannitol The PEG m solution is sufficiently absorbed by gradually adding a portion of the washing medium consisting of Dilute. Add the wash medium (12° and 4.0 mQ) after each dilution. Mix the protoplasts by gentle agitation. Pelletization and regeneration The PEG is removed in a final wash by resuspension in medium.

B. Fusion of protoplasts into protoplasts Preferred protoplast protoplasts The fusion method to the plast involves the fusion of maintenance somatic cells into CMS cells using cell inhibitors. Optimize the Maintains cytoplasmic inhibitory substances in the body. Dobras) 1 Add to part of the suspension . Test different exposures. Iodoacetic acid was tested at a final concentration of 0.1mM. Incubate the cells for 5.10.20 and 30 min at 0°C. preferable The cytoplasmic inhibitor is rhodamine 6-G, which was prepared at 20.40.60 and Test at 1 μg/rnQ for 80 minutes. After exposure is complete, centrifuge The cells are thus pelleted and resuspended in wash medium.

CMS protoplasts treated with 4 levels of X-rays and 4 levels of fine particles Matrix experimental design ( In this example, 16 types of treatments) are used to cover all sensitivity variations.

Tests of suppressed maintainer protoplasts and X-ray treated CMS protoplasts Mix the cells at a 1:1 ratio and add a final sample of 2 x 106 cells in 0.5 mQ of wash medium. Let it be the density. A population with active cells after fusion and no activity before fusion is allogenic It is estimated that it is a group.

In washing medium, 22.5% PEG 6000 (fern?) at pH 5.8-6.0 By adding 2 mQ, 10 mM CaCl2 and 4% sucrose Thus achieving fusion. Incubate cells for 10 minutes at room temperature, then for 5 minutes. 12.5 d of wash medium added at intervals of 0.5, 1.0.2.0.3.0.4.0 mQ Wash to eliminate fusion buffer by diluting with Pelletization and regeneration The fusion buffer is removed in a final wash by suspension in medium.

■ Plating protoplast cultures and regenerating callus The fused protoplasts were grown in regeneration medium to an initial density of 1 x 10 protoplasts)/ Cultivate in mQ. The regeneration medium is 150 m from the basal medium? ／　／　D-Xylo - arabinose, 100 g/l glucose, 34 g/l z Sucrose, 45.5g//Sorbitol, 0.01m9//Dimethylallyl Contains adenine and 0.2/12.4-dichlorophenoxyacetic acid (pH 5,8). Ink the protoplasts for 5-7 days at 25 °C in a dark growth chamber. 26°C and then transferred to low intensity fluorescence at about 26°C. After 3-4 weeks, protopra The insects divide and form small microscopic colonies. Place the colony in basal medium put in. The basal medium is changed twice over a two week period. After 2 weeks, the colony was Plate and remove onto basal agar medium. Embryos and seedlings growing after 1-2 weeks The streaks were isolated and placed on basal agar medium with 0°3/l naphthylene acetic acid for root growth. Culture in tubes.

IX, Growth of differentiated seedlings Remove the mature plants from the tubes and place them in the mist chamber of the greenhouse to cover the soil. Move. Spring break of established plants by placing them in a cold environment (40℃) for 6 to 8 weeks do. Early-flowering and post-spring flowering plants are examined for male sterility, and the male Sexually sterile plants are crossed with maintainer lines in screening cages. by insects to achieve pollination. Harvesting seeds and sowing to determine the stability of newly grown CMS lines. decide.

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Claims (12)

[Claims] 1. Cytoplasmic male sterility of Dei carota with the nucleus of the maintenance cultivar of Daucus carota Demonstrates male sterility characteristics characterized by combining with enucleated protoplasts of cultivars. Method of introgression into maintenance cultivars of I. carota. 2. The isolated nuclei of the maintenance cultivar of Dei carota were isolated from the cytoplasmic male sterile cultivar Dei Carota. 2. The method according to item 1 above, wherein the method is inserted into enucleated protoplasts of data varieties. 3. Maintaining the cytoplasmic male sterile nucleated protoplasts of Dei carota cultivar Dei - The method of item 1 above, which fuses with enucleated protoplasts of the Carota variety. 4. The nucleated protoplasts of the maintainer Dei carota undergo cytoplasmic repression prior to fusion. The method of item 3 above, wherein the method is treated with a control substance. 5. the cytoplasmic inhibitor is selected from the group consisting of iodoacetic acid and rhodamine 6-G; 4. The method of item 4 above. 6. Has a significant amount of cytoplasm from male sterile cultivars and nuclei from maintainer cultivars. Allogenic carrot protoplast lines. 7. Clause 6, wherein said nucleus is selected from the Nantes variety of carrot. carrot protoplast lineage. 8. A cell line regenerated from the protoplast of item 6 above. 9. An allogenic carrot variety regenerated from the line of item 8 above. 10. Consists of cells with cytoplasm of male sterile cultivars and nuclei of maintainer cultivars. Allogenic carrot varieties. 11. Seeds of allogenic carrot varieties comprising cells as described in paragraph 10 above. . 12. One or more crosses containing the parent line produced by the method of paragraph 1 above. Carrot hybrids that arise from miscellaneous.